WO2016187224A1 - Procédés d'amplification d'acides nucléiques et compositions permettant de les mettre en œuvre - Google Patents

Procédés d'amplification d'acides nucléiques et compositions permettant de les mettre en œuvre Download PDF

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WO2016187224A1
WO2016187224A1 PCT/US2016/032919 US2016032919W WO2016187224A1 WO 2016187224 A1 WO2016187224 A1 WO 2016187224A1 US 2016032919 W US2016032919 W US 2016032919W WO 2016187224 A1 WO2016187224 A1 WO 2016187224A1
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copy number
sequencing
nucleic acid
interest
nucleic acids
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Kristen Mary BORCHERT
Frances Poyen TONG
Charlotte Anne BROWN
Richard Lee KELLEY
Chang Chen
Jeffrey Propse BAKER
Stacy XU
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Becton Dickinson and Co
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Becton Dickinson and Co
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2535/00—Reactions characterised by the assay type for determining the identity of a nucleotide base or a sequence of oligonucleotides
    • C12Q2535/122—Massive parallel sequencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2537/00—Reactions characterised by the reaction format or use of a specific feature
    • C12Q2537/10—Reactions characterised by the reaction format or use of a specific feature the purpose or use of
    • C12Q2537/143—Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis

Definitions

  • a microwell containing a template DNA strand to be sequenced is flooded with a single species of deoxyribonucleotide triphosphate (dNTP). If the introduced dNTP is complementary to the leading template nucleotide, it is incorporated into the growing complementary strand. This causes the release of a hydrogen ion that triggers an ISFET ion sensor, which indicates that a reaction has occurred. If homopolymer repeats are present in the template sequence, multiple dNTP molecules will be incorporated in a single cycle. This leads to a corresponding number of released hydrogens and a proportionally higher electronic signal.
  • dNTP deoxyribonucleotide triphosphate
  • Copy number variations are alterations observed in the genome that result in genes having an abnormal number of copies - either more or less than the expected number of two. CNVs may be indicative of large-scale chromosomal rearrangements, such as large insertions or deletions, which can be commonly found in cancer tissue. In some cases, entire chromosomes can be lost or duplicated, which is a common cause of genetic disorders, such as Down syndrome (trisomy 21), cat eye syndrome (trisomy 22), Williams syndrome (monosomy 7), and various others. Identifying copy number variations can help understand and diagnose cancer and aneuploidy-related disorders. CNVs play a large role in cancer and detection of altered numbers of copies of certain genes can provide physicians with information to guide therapy.
  • CNVs have been detected with cytogenetic techniques such as array-based comparative genome hybridization and molecular techniques such as SNP arrays. More recently, detection of CNVs by next- generation sequencing (NGS) has proven feasible, enabling researchers to detect three major categories of genomic alteration - single-nucleotide polymorphisms (SNPs), insertions/deletions, and CNVs in one assay platform.
  • NGS next- generation sequencing
  • the methods include combining a nucleic acid sample and one or more amplification primers adapted to amplify a region of one or more copy number stable genes in a reaction mixture under conditions sufficient to amplify the one or more copy number stable genes.
  • compositions and kits that find use in practicing embodiments of the methods.
  • FIG. 1 provides next generation sequencing (NGS) data obtained using a custom NGS sequencing panel according to one embodiment of the present disclosure.
  • NGS next generation sequencing
  • a sequencing library was prepared using a panel of primers adapted to amplify ten distinct copy number stable genes, as well as genes of interest present in the sample.
  • FIG. 2 graphically illustrates copy number stable regions of the human genome identified using an approach according to one embodiment of the present disclosure.
  • FIG. 3 shows non-limiting examples of genes which may be amplified, e.g., as part of preparing an NGS sequencing library, according to certain aspects of the present disclosure.
  • the methods include combining a nucleic acid sample and one or more amplification primers adapted to amplify a region of one or more copy number stable genes in a reaction mixture under conditions sufficient to amplify the one or more copy number stable genes.
  • compositions and kits that find use in practicing embodiments of the methods.
  • aspects of the present disclosure include methods of amplifying nucleic acids.
  • the methods include combining a nucleic acid sample and one or more amplification primers adapted to amplify a region of one or more copy number stable genes in a reaction mixture under conditions sufficient to amplify the one or more copy number stable genes.
  • aspects of the invention include producing a reaction mixture from a sample and one or more amplification primers adapted to amplify a region of one or more copy number stable genes.
  • copy number stable gene is meant a gene or genomic region that is refractory to duplication or loss (e.g., is refractory to copy number variation (CNV)).
  • CNV copy number variation
  • a copy number stable gene may be a gene or genomic region having a copy number that does not vary (or substantially vary) between a genome of interest and a reference genome.
  • a copy number stable gene has the same copy number in 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% of the genomes in a population of interest, e.g., humans, or human subpopulations of interest (e.g., racial/ethnic human subpopulations, or the like).
  • amplicons produced from copy number stable genes find use, e.g., as internal copy number controls in amplification- and/or sequencing-based assays for determining the copy number (and optionally, the presence of CNVs) in one or more genes of interest in a nucleic acid sample of interest.
  • genes of interest are clinically relevant, e.g., genes for which copy number variation is associated with a disease or disorder, such as cancer, aneuploidy-related disorders (e.g., trisomy 21 , trisomy 22, monosomy 7, etc.), and the like.
  • amplification of the one or more copy number stable genes finds use, e.g., in diagnosing a condition known to be associated with one or more CNVs.
  • the diagnosis includes sequencing (e.g., by NGS sequencing) the amplicons of the one or more copy number stable genes and one or more genes of interest to determine the copy number (and optionally, detect CNV, if any) in the one or more genes of interest.
  • the one or more copy number stable genes are located in ohnolog-rich regions (ORRs) of the genome.
  • An "ohnolog” is a duplicated gene derived from whole genome duplication (WGD).
  • an ORR is a genomic region (e.g., a human genomic region) having a proportion of ohnologs that is 50% or greater, 55% or greater, 60% or greater, 65% or greater, 70% or greater, or 75% or greater in a 2 Mb window.
  • the one or more copy number stable genes include one or more ohnologs. A detailed description of human ohnolog-rich regions and human ohnologs is found in Makino et al.
  • the combining comprises combining a known amount of nucleic acids corresponding to one or more copy number stable genes with the nucleic acid sample and the one or more amplification primers adapted to amplify a region of the nucleic acids corresponding to one or more copy number stable genes.
  • the one or more amplification primers adapted to amplify a region of one or more copy number stable genes may be adapted to amplify any useful copy number stable genes, including any of the copy number stable genes described herein, e.g., ohnologs, genes or genomic regions that are refractory to duplication or loss within ohnolog-rich regions, and the like.
  • Any amplification primer, or combination of two or more amplification primers, adapted to amplify the one or more copy number stable genes (and optionally, one or more nucleic acids of interest) may be employed.
  • the one or more amplification primers are adapted to amplify one, each of, or any combination of the copy number stable genes HNRNPR (Entrez Gene ID 10236), TCEB3 (Entrez Gene ID 6924), IL22RA1 (Entrez Gene ID 58985), RCAN3 (Entrez Gene ID 1 1123), GJB5 (Entrez Gene ID 2707), SLC25A44 (Entrez Gene ID 9673), MT3 (Entrez Gene ID 4504), MT1X (Entrez Gene ID 4501), NUP93 (Entrez Gene ID 9688) and/or RABL2B (Entrez Gene ID 11 158).
  • HNRNPR Entrez Gene ID 10236
  • TCEB3 Entrez Gene ID 6924
  • IL22RA1 Entrez Gene ID 58985
  • RCAN3 Entrez Gene ID 1 1123
  • GJB5 Entrez Gene ID 2707
  • SLC25A44 Entrez Gene ID 9673
  • MT3 Entrez Gene ID 4504
  • CNVs are known to occur in organisms other than humans, such as rodents (e.g., mice and rats), dogs, cattle, rhesus monkeys, chimpanzees, and birds.
  • the one or more amplification primers may be designed/selected to amplify copy number stable genes present in the genome of the relevant non-human organism.
  • a "panel" (or “pool”) of two or more amplification primers is employed. Such pools find use, e.g., when multiplexed amplification of two or more copy number stable genes is desirable.
  • a panel of primers is employed and adapted to amplify 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 or more, 40 or more, 50 or more, or 100 or more copy number stable genes.
  • Such pools may additionally include primers adapted to amplify one or more genes of interest, e.g., genes for which it is desirable to determine copy number information (e.g., for detecting CNV, if any), including but not limited to, genes for which copy number variation is associated with a disease or disorder (e.g., cancer or a particular type thereof, any aneuploidy-related disorder, etc.).
  • genes for which it is desirable to determine copy number information e.g., for detecting CNV, if any
  • a disease or disorder e.g., cancer or a particular type thereof, any aneuploidy-related disorder, etc.
  • a panel of primers may be employed that is adapted to amplify any desirable number of copy number stable genes, and additionally, adapted to amplify any desirable number of genes of interest, including but not limited to, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 or more, 40 or more, 50 or more, 100 or more, 150 or more, 200 or more, 250 or more, 500 or more, or 1000 or more genes of interest.
  • genes of interest finds use, e.g., for SNP genotyping/variant detection by sequencing, genomic profiling, expression profiling, and/or the like.
  • genes of interest which may be amplified according to the methods of the present disclosure include one, each, or any combination of the genes provided in FIG. 3.
  • a panel of amplification primers is employed, and the panel is adapted, in addition to amplifying one or more copy number stable genes, to amplify two or more regions of interest present in genomic DNA, including but not limited to, "hot spot" regions that are frequently mutated in human cancer genes.
  • a panel may be specifically designed by one practicing the subject methods, or the practitioner may order one of the various commercially available panels, such as an Ion AmpliSeqTM Cancer Hotspot Panel available from Life Technologies, Inc. (Carlsbad, CA).
  • the one or more amplification primers may be designed to be sufficiently complementary to their corresponding target nucleic acids in the nucleic acid sample, such that the primer specifically hybridizes to its target under hybridization conditions.
  • complementary refers to a nucleotide sequence that base- pairs by non-covalent bonds to a region of the copy number stable gene or nucleic acid of interest.
  • adenine (A) forms a base pair with thymine (T), as does guanine (G) with cytosine (C) in DNA.
  • thymine is replaced by uracil (U).
  • U uracil
  • A is complementary to T and G is complementary to C.
  • RNA is complementary to U and vice versa.
  • complementary refers to a nucleotide sequence that is at least partially complementary.
  • a nucleotide sequence may be partially complementary to a target, in which not all nucleotides are complementary to every nucleotide in the target nucleic acid in all the corresponding positions.
  • the amplification primer may be perfectly (i.e., 100%) complementary to the copy number stable gene, or the primer and the copy number stable gene may share some degree of complementarity which is less than perfect (e.g., 70%, 75%, 85%, 90%, 95%, 99%).
  • a non-limiting example of such a mathematical algorithm is described in Karlin et al., Proc. Natl. Acad. Sci. USA 90:5873-5877 (1993).
  • NBLAST and XBLAST programs version 2.0 as described in Altschul et al., Nucleic Acids Res. 25:389-3402 (1997).
  • NBLAST BLAST and Gapped BLAST programs
  • hybridization conditions means conditions in which a primer specifically hybridizes to a region of a copy number stable gene or nucleic acid of interest. Whether a primer specifically hybridizes to a target nucleic acid is determined by such factors as the degree of complementarity between the polymer and the target nucleic acid and the temperature at which the hybridization occurs, which may be informed by the melting temperature (T M ) of the primer.
  • T M melting temperature
  • the melting temperature refers to the temperature at which half of the primer-target nucleic acid duplexes remain hybridized and half of the duplexes dissociate into single strands.
  • nucleic acid sequences present in the genomes, transcriptomes, etc. of nucleic acid sources of interest are readily available from resources such as the nucleic acid sequence databases of the National Center for Biotechnology Information (NCBI), the European Molecular Biology Laboratory-European Bioinformatics Institute (EMBL-EBI), and the like. Based on such sequence information, one can design/select one or more amplification primers to amplify the one or more copy number stable genes, and optionally, one or more nucleic acids of interest.
  • NCBI National Center for Biotechnology Information
  • EBL-EBI European Molecular Biology Laboratory-European Bioinformatics Institute
  • the one or more amplification primers include a sequencing adapter
  • sequencing adapter is meant one or more nucleic acid domains that include at least a portion of a nucleic acid sequence (or complement thereof) utilized by a sequencing platform of interest, such as a sequencing platform provided by lllumina® (e.g., the HiSeqTM, MiSeqTM and/or Genome AnalyzerTM sequencing systems); Ion TorrentTM (e.g., the Ion PGMTM and/or Ion ProtonTM sequencing systems); Pacific Biosciences (e.g., the PACBIO RS II sequencing system); Life TechnologiesTM (e.g., a SOLiD sequencing system); Roche (e.g., the 454 GS FLX+ and/or GS Junior sequencing systems); or any other sequencing platform of interest.
  • lllumina® e.g., the HiSeqTM, MiSeqTM and/or Genome AnalyzerTM sequencing systems
  • Ion TorrentTM e.g., the Ion PGMTM and/or Ion ProtonTM sequencing systems
  • the one or more amplification primers include a sequencing adapter that includes a nucleic acid domain selected from: a domain (e.g., a "capture site” or “capture sequence”) that specifically binds to a surface-attached sequencing platform oligonucleotide (e.g., the P5 or P7 oligonucleotides attached to the surface of a flow cell in an lllumina® sequencing system); a sequencing primer binding domain (e.g., a domain to which the Read 1 or Read 2 primers of the lllumina® platform may bind); a barcode domain (e.g., a domain that uniquely identifies the sample source of the nucleic acid being sequenced to enable sample multiplexing by marking every molecule from a given sample with a specific barcode or "tag”); a barcode sequencing primer binding domain (a domain to which a primer used for sequencing a barcode binds); a molecular identification domain (e.g., a molecular index tag
  • a barcode domain e.g., sample index tag
  • a molecular identification domain e.g., a molecular index tag
  • the one or more amplification primers may include a sequencing adapter of any length and sequence suitable for the sequencing platform of interest.
  • the nucleic acid domains are from 4 to 100 nucleotides in length, such as from 6 to 75, from 8 to 50, or from 10 to 40 nucleotides in length.
  • the one or more amplification primers may include one or more nucleotides (or analogs thereof) that are modified or otherwise non-naturally occurring.
  • the amplification primers may include one or more nucleotide analogs (e.g., LNA, FANA, 2'-0-Me RNA, 2'-fluoro RNA, or the like), linkage modifications (e.g., phosphorothioates, 3'-3' and 5'-5' reversed linkages), 5' and/or 3' end modifications (e.g., 5' and/or 3' amino, biotin, DIG, phosphate, thiol, dyes, quenchers, etc.), one or more fluorescently labeled nucleotides, or any other feature that provides a desired functionality to the primers and/or resulting amplicons.
  • nucleotide analogs e.g., LNA, FANA, 2'-0-Me RNA, 2'-fluoro RNA, or the like
  • the nucleic acid sample may be any nucleic acid sample that includes, or is suspected of including, one or more copy number stable genes, and optionally, one or more nucleic acids of interest, e.g., one or more nucleic acids in addition to the one or more copy number stable genes for which amplification is desirable.
  • Amplification of one or more copy number stable genes and one or more nucleic acids of interest may be desirable for a variety of reasons, including but not limited to, sequencing the amplification products (or "amplicons") of the one or more copy number stable genes and one or more nucleic acids of interest.
  • Sequencing the amplicons enables one to determine the nucleotide sequence(s) of - and number of sequencing runs corresponding to - the one or more copy number stable genes and one or more nucleic acids of interest, which may in turn be used to determine the copy number of one or more of the nucleic acids of interest as described in detail below.
  • determining the copy number of one or more of the nucleic acids of interest enables one to determine whether copy number variation (CNV) exists for one or more of the one or more nucleic acids of interest in the sample.
  • CNV copy number variation
  • the nucleic acid sample is isolated from a source other than a mammal, such as bacteria, yeast, insects (e.g., drosophila), amphibians (e.g., frogs (e.g., Xenopus)), viruses, plants, or any other non-mammalian nucleic acid sample source.
  • a source other than a mammal such as bacteria, yeast, insects (e.g., drosophila), amphibians (e.g., frogs (e.g., Xenopus)), viruses, plants, or any other non-mammalian nucleic acid sample source.
  • the nucleic acid sample is isolated from a biological sample, such as a biological fluid or a biological tissue.
  • biological fluids include urine, blood, plasma, serum, saliva, semen, stool, sputum, cerebral spinal fluid, tears, mucus, sperm, amniotic fluid or the like.
  • Biological tissues are aggregates of cells, usually of a particular kind together with their intercellular substance that form one of the structural materials of a human, animal, plant, bacterial, fungal or viral structure, including connective, epithelium, muscle and nerve tissues. Examples of biological tissues also include organs, tumors, lymph nodes, arteries and individual cells.
  • the nucleic acid sample is a tumor nucleic acid sample (that is, a nucleic acid sample isolated from a tumor).
  • Tumor refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.
  • cancer and “cancerous” refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth/proliferation. Examples of cancer include but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia.
  • the nucleic acid sample includes nucleic acids from one or more circulating tumor cells (CTCs).
  • CTCs circulating tumor cells
  • the nucleic acid sample is a deoxyribonucleic acid (DNA) sample.
  • DNA samples of interest include, but are not limited to, genomic DNA samples, mitochondrial DNA samples, complementary DNA (cDNA, synthesized from any RNA or DNA of interest) samples, recombinant DNA samples (e.g., plasmid DNA samples), and any other DNA samples of interest.
  • the nucleic acid sample is a ribonucleic acid (RNA) sample.
  • RNA samples of interest include, but are not limited to, messenger RNA (mRNA) samples, small/short interfering RNA (siRNA) samples, microRNA (miRNA) samples, any other DNA samples of interest.
  • mRNA samples messenger RNA samples
  • siRNA samples small/short interfering RNA samples
  • miRNA samples microRNA samples
  • Approaches, reagents and kits for isolating DNA and RNA from sources of interest are known in the art and commercially available.
  • kits for isolating DNA from a source of interest include the DNeasy®, RNeasy®, QIAamp®, QIAprep® and QIAquick® nucleic acid isolation/purification kits by Qiagen, Inc.
  • the nucleic acid is isolated from a fixed biological sample, e.g., formalin-fixed, paraffin- embedded (FFPE) tissue. Genomic DNA and RNA from FFPE tissue may be isolated using commercially available kits - such as the AHPrep® DNA/RNA FFPE kit by Qiagen, Inc.
  • FFPE formalin-fixed, paraffin- embedded
  • the sample may be subjected to shearing/fragmentation, e.g., to generate nucleic acids that are shorter in length as compared to precursor non-sheared nucleic acids (e.g., genomic DNA) in the original sample.
  • shearing/fragmentation strategies include, but are not limited to, passing the sample one or more times through a micropipette tip or fine-gauge needle, nebulizing the sample, sonicating the sample (e.g., using a focused-ultrasonicator by Covaris, Inc.
  • bead-mediated shearing e.g., using one or more DNA-shearing e.g., restriction, enzymes
  • enzymatic shearing e.g., using one or more DNA-shearing e.g., restriction, enzymes
  • chemical based fragmentation e.g., using divalent cations
  • fragmentation buffer which may be used in combination with heat
  • the nucleic acids generated by shearing/fragmentation of a starting nucleic acid sample has a length of from 50 to 10,000 nucleotides, from 100 to 5000 nucleotides, from 150 to 2500 nucleotides, from 200 to 1000 nucleotides, e.g., from 250 to 500 nucleotides in length.
  • the nucleic acids generated by shearing/fragmentation of a starting nucleic acid sample has a length of from 10 to 20 nucleotides, from 20 to 30 nucleotides, from 30 to 40 nucleotides, from 40 to 50 nucleotides, from 50 to 60 nucleotides, from 60 to 70 nucleotides, from 70 to 80 nucleotides, from 80 to 90 nucleotides, from 90 to 100 nucleotides, from 100 to 150 nucleotides, from 150 to 200, from 200 to 250 nucleotides in length, or from 200 to 1000 nucleotides or even from 1000 to 10,000 nucleotides, for example, as appropriate for a sequencing platform in which one desires to sequence amplicons produced upon amplification of the one or more copy number stable genes and any other amplicons that may be present, e.g., amplicons produced from nucleic acids of interest present in the nucleic acid sample.
  • the nucleic acid sample and the one or more amplification primers adapted to amplify a region of one or more copy number stable genes are combined in a reaction mixture under conditions sufficient to amplify the one or more copy number stable genes.
  • condition sufficient to amplify the one or more copy number stable genes is meant reaction conditions that permit polymerase-mediated extension of a 3' end of the one or more amplification primers.
  • Achieving suitable reaction conditions may include selecting reaction mixture components, concentrations thereof, and a reaction temperature to create an environment in which a polymerase is active and the relevant nucleic acids in the reaction interact (e.g., hybridize) with one another in the desired manner. Suitable hybridization conditions are described in detail above.
  • the reaction mixture may include buffer components that establish an appropriate pH, salt concentration (e.g., KCI concentration), metal cofactor concentration (e.g., Mg 2+ or Mn 2+ concentration), and the like, for the extension reaction to occur.
  • salt concentration e.g., KCI concentration
  • metal cofactor concentration e.g., Mg 2+ or Mn 2+ concentration
  • nuclease inhibitors e.g., a DNase inhibitor and/or an RNase inhibitor
  • additives for facilitating amplification/replication of GC rich sequences e.g., one or more additives for facilitating amplification/replication of GC rich sequences
  • enzyme-stabilizing components e.g., DTT present at a final concentration ranging from 1 to 10 mM (e.g., 5 rtiM)
  • any other reaction mixture components useful for facilitating polymerase-mediated extension reactions.
  • the reaction mixture can have a pH suitable for the primer extension reaction.
  • the pH of the reaction mixture ranges from 5 to 9, such as from 7 to 9.
  • the reaction mixture includes a pH adjusting agent.
  • pH adjusting agents of interest include, but are not limited to, sodium hydroxide, hydrochloric acid, phosphoric acid buffer solution, citric acid buffer solution, and the like.
  • the pH of the reaction mixture can be adjusted to the desired range by adding an appropriate amount of the pH adjusting agent.
  • the temperature range suitable for amplification may vary according to factors such as the particular polymerase employed, the melting temperatures of the one or more amplification primers employed, etc.
  • the reaction mixture conditions include bringing the reaction mixture to a temperature ranging from 4° C to 80° C, such as from 16° C to 75° C, e.g., from 37° C to 72° C.
  • the methods of the present disclosure may include one or more steps in addition to the combining step described above.
  • the methods may further include utilizing the amplified one or more copy number stable genes (and any other amplicons that may be present) in a downstream application/assay of interest.
  • the amplified nucleic acids may be utilized directly (optionally after a purification step), or may be modified prior to being utilized in a downstream application/assay of interest.
  • the methods further include adding a sequencing adapter to the amplified one or more copy number stable genes and any other amplicons that may be present. Such a step may be performed whether or not the amplicons already include one or more sequencing adapters (e.g., by virtue of the one or more amplification primers including one or more sequencing adapters as described above).
  • Sequencing adapters that may be added include, e.g., one or more capture domains, one or more sequencing primer binding domains, one or more barcode domains, one or more barcode sequencing primer binding domains, one or more molecular identification domains, a complement of any such domains, or any combination thereof. Further details regarding sequencing adapters are described hereinabove.
  • the methods include sequencing the amplified one or more copy number stable genes, and any other amplicons produced during the combining step, e.g., amplicons produced from one or more nucleic acids of interest as described above.
  • amplification products may be sequenced directly (optionally after a purification step), or may be modified prior to being sequenced. Modifications prior to sequencing include, but are not limited to, the addition of one or more sequencing adapters as described above, and/or any other useful modifications for sequencing the amplicons on a sequencing platform of interest.
  • the sequencing may be carried out on any suitable sequencing platform, including a Sanger sequencing platform, a next generation sequencing (NGS) platform (e.g., using a next generation sequencing protocol), or the like.
  • NGS sequencing platforms of interest include, but are not limited to, a sequencing platform provided by lllumina® (e.g., the HiSeqTM, MiSeqTM and/or Genome AnalyzerTM sequencing systems); Ion TorrentTM (e.g., the Ion PGMTM and/or Ion ProtonTM sequencing systems); Pacific Biosciences (e.g., the PACBIO RS II sequencing system); Life TechnologiesTM (e.g., a SOLiD sequencing system); Roche (e.g., the 454 GS FLX+ and/or GS Junior sequencing systems); or any other sequencing platform of interest.
  • lllumina® e.g., the HiSeqTM, MiSeqTM and/or Genome AnalyzerTM sequencing systems
  • Ion TorrentTM e.g., the I
  • the methods further include determining the copy number of the one or more nucleic acids of interest present in the nucleic acid sample (e.g., the copy number of a nucleic acid of interest present in a genome from which the nucleic acid sample is derived. Such a determination may be based on, e.g., the number of sequencing reads corresponding to the one or more nucleic acids of interest present in the nucleic acid sample, and the number of sequencing reads corresponding to the one or more copy number stable genes.
  • determining the copy number of the one or more nucleic acids of interest present in the nucleic acid sample includes determining a ratio of the number of sequencing reads corresponding to the one or more nucleic acids of interest present in the nucleic acid sample to the number of sequencing reads corresponding to the one or more copy number stable genes. Determining the copy number of the one or more nucleic acids of interest present in the nucleic acid sample may be based on a ratio.
  • the following formula is used to determine the copy number of a nucleic acid of interest present in a genome from which the nucleic acid sample is derived:
  • c the copy number
  • r the number of sequencing reads
  • NA the nucleic acid of interest
  • CNSG the copy number stable gene.
  • the methods of the present disclosure find use in a variety of applications, including but not limited to, applications in which it is desirable to determine the copy number of one or more genomic regions (e.g., genes, intergenic regions, etc.) present in a source of nucleic acids (e.g., cells or tissues of interest, such as tumor cells or tissues of interest).
  • Applications of interest include, e.g., research applications, clinical applications (e.g., clinical diagnostic applications), etc., and the methods may be employed in such applications to assess whether, e.g., a cell or tissue exhibits copy number variation (CNV) in one or more genomic regions of interest.
  • the methods also find use in determining the nucleotide sequences of nucleic acids amplified from the nucleic acid sample and/or quantifying the amount of the one or more nucleic acids of interest present in the sample.
  • the methods of the present disclosure which involve the amplification of one or more copy number stable genes - provide advantages over existing approaches in a number of respects.
  • the methods of the present disclosure are advantageous in the context of nucleic acid sequencing for reasons including, but not limited to, the presence of internal control standards in sequencing libraries generated using the methods of the present disclosure, which internal control standards enable sample normalization and accurate copy number determination.
  • the internal control standards are the amplicons produced from the one or more copy number stable genes using the method of the present disclosure.
  • the amplicons produced from the one or more copy number stable genes may be used to normalize sequencing reads so that copy number information may be derived from sequencing data, obviating the need for matched samples (e.g., matched tumor/normal samples), pooled normal controls, universal control standards, and/or the like.
  • matched samples e.g., matched tumor/normal samples
  • pooled normal controls e.g., pooled normal controls
  • universal control standards e.g., the number of patient samples that can be analyzed in a given sequencing run.
  • the provision of internal copy number controls as provided by embodiments of the present disclosure therefore, increases sample throughput relative to current approaches.
  • the methods provide sequencing read normalization required for NGS sequencing Copy Number Variation (CNV) determination that is built into each sample's sequencing library preparation reaction, obviating the requirement for additional control samples, each control sample requiring a separate library preparation reaction, sequencing run, etc.
  • CNV NGS sequencing Copy Number Variation
  • compositions of the present disclosure further include compositions.
  • the compositions of the present disclosure find a variety of uses, including in some aspects, practicing the methods of the present disclosure.
  • composition that includes a nucleic acid sample and one or more amplification primers adapted to amplify a region of one or more copy number stable genes.
  • the composition may include any nucleic acid sample of interest and any suitable amplification primer(s), including any of the nucleic acid samples and amplification primers described above in the section relating to the methods of the present disclosure.
  • the composition includes one or more amplification primers adapted to amplify one or more nucleic acids of interest present in the nucleic acid sample, in addition to the one or more amplification primers adapted to amplify a region of one or more copy number stable genes.
  • the composition may include a pool (or "panel") of primers for amplification of one or more nucleic acids of interest and one or more copy number stable genes.
  • Panels of interest include, but are not limited to, sequencing panels adapted to amplify one or more genomic regions (e.g., genes) of interest in a tissue of interest (e.g., tumor tissue) and one or more copy number stable genes. Downstream sequencing of the copy number stable genes enables, e.g., determination of copy number and any variation thereof in the one or more genomic regions of interest.
  • compositions of the present disclosure include, but are not limited to, a polymerase, dNTPs, a buffer component that establishes an appropriate pH, a salt (e.g., e.g., NaCI, KCI, or the like), a metal cofactor (e.g., Mg 2+ , Mn 2+ , or the like), a nuclease inhibitor (e.g., a DNase inhibitor and/or an RNase inhibitor), an additive for facilitating amplification/replication of GC rich sequences, an enzyme-stabilizing component (e.g., DTT), any other reaction mixture components (e.g., useful for facilitating polymerase- mediated extension reactions), a known amount of internal standard nucleic acids corresponding to one or more copy number stable genes, and any combination thereof.
  • a salt e.g., e.g., NaCI, KCI, or the like
  • a metal cofactor e.g., Mg 2+ , Mn 2+ , or the
  • composition of the present disclosure includes the amplicons produced by the methods of the present disclosure.
  • such compositions include the amplicons in purified form (e.g., substantially or completely separated from the amplification reaction mixture components).
  • the amplicons may include a sequencing adapter provided during or after the amplification reaction as described above.
  • compositions of the present disclosure may be present in a container.
  • suitable containers include, but are not limited to, tubes, vials, plates (e.g., a 96- or other-well plate).
  • compositions of the present disclosure may be present in a device.
  • Devices of interest include, but are not limited to, an incubator, a thermocycler, a sequencing system (e.g., a Sanger sequencing system or a next generation sequencing system), a microfluidic device, or the like.
  • nucleic acid sequencing systems find use in sequencing amplicons generated using the methods of the present disclosure.
  • a sequencing system of the present disclosure includes a collection of nucleic acids.
  • the collection of nucleic acids includes amplicons corresponding to nucleic acids of interest present in a nucleic acid sample, and amplicons corresponding to one or more copy number stable genes present in the nucleic acid sample.
  • the sequencing system includes amplicons generated from any of the one or more amplification primers adapted to amplify a region of one or more copy number stable genes and any of the one or more amplification primers adapted to amplify one or more nucleic acids of interest as described above in the section relating to the methods of the present disclosure.
  • the amplicons may include a sequencing adapter provided during the amplification reaction that produced the amplicons (e.g., provided according to embodiments of the subject methods) and/or after the amplification reaction (e.g., provided according to embodiments of the subject methods).
  • a sequencing adapter provided during the amplification reaction that produced the amplicons (e.g., provided according to embodiments of the subject methods) and/or after the amplification reaction (e.g., provided according to embodiments of the subject methods).
  • the sequencing system may be any sequencing system of interest, including a Sanger sequencing system, a next generation sequencing (NGS) system, or the like.
  • the sequencing system is an NGS system.
  • NGS systems of interest include, but are not limited to, a sequencing system provided by lllumina® (e.g., the HiSeqTM, MiSeqTM and/or Genome AnalyzerTM sequencing systems); Ion TorrentTM (e.g., the Ion PGMTM and/or Ion ProtonTM sequencing systems); Pacific Biosciences (e.g., the PACBIO RS II sequencing system); Life TechnologiesTM (e.g., a SOLiD sequencing system); Roche (e.g., the 454 GS FLX+ and/or GS Junior sequencing systems), or any other suitable NGS systems.
  • lllumina® e.g., the HiSeqTM, MiSeqTM and/or Genome AnalyzerTM sequencing systems
  • Ion TorrentTM e.g., the Ion PGMTM
  • the collection of nucleic acids may be present in a component of the sequencing system.
  • the collection of nucleic acids may be present in a sample preparation component of the sequencing system, e.g., a component of the sequencing system where nucleic acids of the collection are fragmented and/or sequencing adapters are added to the nucleic acids of the collection.
  • the collection of nucleic acids may be present in a solid-phase amplification component of the sequencing system, where solid- phase amplification of the nucleic acids of the collection may occur.
  • An example of such a solid-phase amplification component of a sequencing system is the flow cell of lllumina-based sequencing systems, where cluster generation occurs.
  • a solid-phase amplification component of a sequencing system is the Ion OneTouchTM 2 component for producing templates suitable for sequencing on an Ion PGMTM system, Ion ProtonTM system, or other NGS system provided by Ion TorrentTM.
  • the collection of nucleic acids may be present in any component of a sequencing system useful for utilizing the collection of nucleic acids to obtain the nucleic acid sequences thereof.
  • the sequencing system is adapted to determine the copy number of the nucleic acids of interest present in the nucleic acid sample. In certain aspects, the determination is based on the number of sequencing reads corresponding to nucleic acids of interest in the nucleic acid sample, and the number of sequencing reads corresponding to the one or more copy number stable genes. In certain aspects, such a sequencing system is adapted to determine a ratio of the number of sequencing reads corresponding to nucleic acids of interest in the nucleic acid sample to the number of sequencing reads corresponding to the one or more copy number stable genes.
  • the system may be further adapted to determine the copy number of the nucleic acids of interest present in the nucleic acid sample based on the ratio of the number of sequencing reads corresponding to nucleic acids of interest in the nucleic acid sample to the number of sequencing reads corresponding to the one or more copy number stable genes.
  • the sequencing system includes the components and functionality to perform the recited determinations.
  • the sequencing system includes a processor and a computer-readable medium (e.g., a non-transitory computer-readable medium).
  • the computer-readable medium includes instructions executable by the processor to, e.g., determine the copy number of the nucleic acids of interest present in the nucleic acid sample as described above, determine a ratio of the number of sequencing reads corresponding to nucleic acids of interest in the nucleic acid sample to the number of sequencing reads corresponding to the one or more copy number stable genes, and/or the like.
  • Example formulas/algorithms which may be implemented by the sequencing systems of the present disclosure are described above in the section relating to the methods of the present disclosure.
  • kits include one or more amplification primers adapted to amplify a region of one or more copy number stable genes present in a nucleic acid sample of interest, and a container (e.g., a tube).
  • a container e.g., a tube
  • the one or more amplification primers are present in the container.
  • the subject kits may include any of the amplification primers adapted to amplify any of the copy number stable genes described above in relation to the methods of the present disclosure.
  • kits include one or more amplification primers adapted to amplify one or more nucleic acids of interest present in the nucleic acid sample, in addition to the one or more amplification primers adapted to amplify a region of one or more copy number stable genes.
  • a kit of the present disclosure may include a pool (or "panel") of primers for amplification of one or more nucleic acids of interest and one or more copy number stable genes.
  • Panels of interest include, but are not limited to, sequencing panels adapted to amplify one or more genomic regions (e.g., genes) of interest in a tissue of interest (e.g., tumor tissue) and one or more copy number stable genes.
  • kits include one or more of a polymerase, dNTPs, a buffer component that establishes an appropriate pH, a salt (e.g., e.g., NaCI, KCI, or the like), a metal cofactor (e.g., Mg 2+ , Mn 2+ , or the like), a nuclease inhibitor (e.g., a DNase inhibitor and/or an RNase inhibitor), an additive for facilitating amplification/replication of GC rich sequences, an enzyme-stabilizing component (e.g., DTT), internal standard nucleic acids corresponding to one or more copy number stable genes, and/or any other reaction mixture components, e.g., useful for facilitating polymerase-mediated extension reactions.
  • a salt e.g., e.g., NaCI, KCI, or the like
  • a metal cofactor e.g., Mg 2+ , Mn 2+ , or the like
  • a nuclease inhibitor e.g
  • Components of the subject kits may be present in separate containers, or multiple components may be present in a single container.
  • each of the two or more amplification primers may be present in separate containers, subsets of the two or more amplification primers may be present in separate containers, each of the two or more amplification primers may be present in a single container, etc.
  • the one or more amplification primers may be provided in any suitable container.
  • the amplification primers may be provided in a single tube (e.g., vial), in one or more wells of a plate (e.g., a 96-well plate, a 384-well plate, etc.), or the like.
  • kits of the present disclosure may further include instructions for using the components of the kit, e.g., to practice the methods of the present disclosure.
  • the kit may include instructions for using the one or more amplification primers adapted to amplify a region of one or more copy number stable genes to determine the copy number of one or more genes of interest present in the nucleic acid sample of interest.
  • the instructions may be recorded on a suitable recording medium.
  • the instructions may be printed on a substrate, such as paper or plastic, etc.
  • the instructions may be present in the kits as a package insert, in the labeling of the container of the kit or components thereof (i.e., associated with the packaging or sub-packaging) etc.
  • the instructions are present as an electronic storage data file present on a suitable computer readable storage medium, e.g., portable flash drive, DVD, CD-ROM, diskette, etc.
  • the actual instructions are not present in the kit, but means for obtaining the instructions from a remote source, e.g. via the internet, are provided.
  • An example of this embodiment is a kit that includes a web address where the instructions can be viewed and/or from which the instructions can be downloaded.
  • the means for obtaining the instructions is recorded on a suitable substrate.
  • Example 1 Inclusion of Primers Adapted to Amplify Copy Number Stable Genes in a Sequencing Panel
  • amplification primers adapted to amplify 10 genes identified based upon their proximity to ohnologs were included in a sequencing panel for sequencing library preparation.
  • Ohnologs and genes in close proximity to ohnologs have fewer copy number alterations than genes further away from ohnolog-rich regions. See, e.g., Makino et al. (2013) Nature Communications 4:2283.
  • These ten additional genes were included in a custom TumorSeq DNA Panel and served as internal controls for CNV detection.
  • the ten genes included in the panel for the purposes of internal control standards were: HNRNPR (Entrez Gene ID 10236), TCEB3 (Entrez Gene ID 6924), IL22RA1 (Entrez Gene ID 58985), RCAN3 (Entrez Gene ID 11123), GJB5 (Entrez Gene ID 2707), SLC25A44 (Entrez Gene ID 9673), MT3 (Entrez Gene ID 4504), MT1X (Entrez Gene ID 4501), NUP93 (Entrez Gene ID 9688) and RABL2B (Entrez Gene ID 11158). Sequencing data was collected using a custom NGS panel that included these ten genes. Data for these ten genes is shown in FIG. 1.
  • the ten genes identified to serve as internal controls were identified using both the reference mentioned and bioinformatics tools, using the following method: (1) regions on chromosomes lacking ClinVar and DGV entries for CNV were located using bioinformatics tools; (2) coordinates from supplementary Table 1 of Makino et al. (supra) were updated to reflect the current build of the human genome (hg19), using bioinformatics tools; (3) CNV-free regions identified in step (1) were cross-referenced with the table from step (2); (4) genes closest to ohnologs were identified; and (5) CNV status was confirmed using DGV (Database of Genomic Variation) entry data from Gene Cards. Copy number stable genes in the human genome identified using this approach are graphically illustrated in FIG. 2.
  • a method of amplifying nucleic acids comprising:
  • one or more amplification primers adapted to amplify a region of one or more copy number stable genes, in a reaction mixture under conditions sufficient to amplify the one or more copy number stable genes.
  • the one or more amplification primers are adapted to amplify a region of one or more copy number stable genes selected from the group consisting of: HNRNPR, TCEB3, IL22RA1 , RCAN3, GJB5, SLC25A44, MT3, MT1X, NUP93, RABL2B, and combinations thereof.
  • IL22RA1 IL22RA1 , RCAN3, GJB5, SLC25A44, MT3, MT1X, NUP93, and RABL2B.
  • nucleic acid sample comprises nucleic acids isolated from one or more cells of a cellular sample of interest.
  • determining the copy number of the one or more nucleic acids of interest present in the nucleic acid sample is based on a ratio of the number of sequencing reads corresponding to the one or more nucleic acids of interest present in the nucleic acid sample to the number of sequencing reads corresponding to the one or more copy number stable genes.
  • composition comprising:
  • one or more amplification primers adapted to amplify a region of one or more copy number stable genes.
  • composition according to Clause 19 wherein the one or more amplification primers are adapted to amplify one or more copy number stable genes located in ohnolog-rich regions of a genome.
  • composition according to Clause 20 wherein the ohnolog-rich regions of the genome are regions comprising a proportion of ohnologs greater than 50% in a 2 Mb window.
  • composition according to any one of Clauses 19 to 21 wherein the one or more amplification primers are adapted to amplify one or more ohnologs.
  • composition according to Clause 23 wherein the one or more amplification primers are adapted to amplify a region of each of the copy number stable genes HNRNPR, TCEB3, IL22RA1 , RCAN3, GJB5, SLC25A44, MT3, MT1X, NUP93, and RABL2B. 25.
  • composition according to Clause 26 or Clause 27, wherein the cellular sample of interest is a tumor sample.
  • nucleic acids comprising: amplicons corresponding to nucleic acids of interest present in a nucleic acid sample; and amplicons corresponding to one or more copy number stable genes.
  • GJB5 SLC25A44, MT3, MT1X, NUP93, RABL2B, and combinations thereof.
  • nucleic acid sample comprises nucleic acids isolated from one or more cells of a cellular sample of interest.
  • sequencing system according to any one of Clauses 31 to 41 , wherein the sequencing system is adapted to determine the copy number of the nucleic acids of interest present in the nucleic acid sample based on: the number of sequencing reads corresponding to nucleic acids of interest in the nucleic acid sample; and the number of sequencing reads corresponding to the one or more copy number stable genes.
  • sequencing system according to Clause 42, wherein the sequencing system is adapted to determine the copy number of the nucleic acids of interest present in the nucleic acid sample based on a ratio of the number of sequencing reads corresponding to nucleic acids of interest in the nucleic acid sample to the number of sequencing reads corresponding to the one or more copy number stable genes.
  • a kit comprising:
  • one or more amplification primers adapted to amplify a region of one or more copy number stable genes present in a nucleic acid sample of interest
  • kits according to Clause 45 wherein the one or more amplification primers are adapted to amplify one or more copy number stable genes located in ohnolog-rich regions of a genome.
  • kits according to Clause 46 wherein the ohnolog-rich regions of the genome are regions comprising a proportion of ohnologs greater than 50% in a 2 Mb window.
  • kits according to Clause 46 wherein the one or more amplification primers are adapted to amplify a region of one or more copy number stable genes selected from the group consisting of: HNRNPR, TCEB3, IL22RA1 , RCAN3, GJB5, SLC25A44, MT3, MT1X, NUP93, RABL2B, and combinations thereof.
  • nucleic acid sample comprises nucleic acids isolated from one or more cells of a cellular sample of interest.
  • kits according to any one of Clauses 45 to 54, wherein the one or more amplification primers comprise a sequencing adapter.
  • kit according to any one of Clauses 45 to 56 further comprising instructions for using the one or more amplification primers adapted to amplify a region of one or more copy number stable genes to determine the copy number of one or more genes of interest present in the nucleic acid sample of interest.

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Abstract

La présente invention concerne des procédés d'amplification d'acides nucléiques. Les procédés consistant à combiner un échantillon d'acide nucléique et une ou plusieurs amorces d'amplification adaptées pour amplifier une région d'un ou de plusieurs gènes stables du nombre de copies dans un mélange réactionnel dans des conditions suffisantes pour amplifier lesdits gènes stables du nombre de copies. Des aspects de la présente invention comprennent en outre des compositions et des kits qui trouvent une utilisation dans la mise en œuvre des modes de réalisation desdits procédés.
PCT/US2016/032919 2015-05-21 2016-05-17 Procédés d'amplification d'acides nucléiques et compositions permettant de les mettre en œuvre Ceased WO2016187224A1 (fr)

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